Cd4+cells with cytolytic properties

ABSTRACT

The present invention relates to CD4+ T cells, more specifically cytolytic or cytotoxic CD4+ T-cells and methods of obtaining and identifying them.

FIELD OF THE INVENTION

The present invention relates to CD4+ T cells, more specificallycytolytic or cytotoxic CD4+ T-cells and methods of obtaining andidentifying them.

BACKGROUND OF THE INVENTION

Natural regulatory T cells (Tregs) are actively selected in the thymusand exert potent suppressive activity in the periphery for the inductionand maintenance of tolerance. These cells are characterised by adistinct phenotype including high expression of cell surface proteinsCD25 and GITR, high intracellular expression of CTLA-4, but absence ofIL-7R. They are anergic and hyporesponsive in the absence of exogenousgrowth factors, and do not produce IL-2. Expression of the transcriptionrepressor Foxp3 is the hallmark of such natural Tregs. The suppressiveactivity of natural Tregs was shown to be linked to a defect inphosphorylation of AKT, a serine-threonine kinase dependent ofphosphatidylinositide-3 kinase (PI3K; Crellin et al. (2007) Blood 109:2014-2022).

The use of such natural Tregs in controlling immune disorders byadoptive cell transfer is severely limited by the very low frequency ofcells of defined specificity, the difficulty to expand them in vitro andby the absence of efficient methods by which they can be expanded invivo. Besides, the functional activity of natural regulatory T cells isnon-specific, as they produce suppressive cytokines such as IL-10 andTGF-beta. Hence, there is a need for suppressor T cells with increasedspecificity that are in addition more amenable to expansion.

SUMMARY OF THE INVENTION

In one aspect, the current invention encompasses isolated populations ofcytotoxic CD4+ T-cells (in either resting or activated state)characterised, when compared to natural CD4+ regulatory T-cells, byabsence of expression or undetectable expression of the transcriptionrepressor Foxp3. In a further embodiment, not excluding the previousembodiment, said population of cytotoxic CD4+ T-cells, in activatedstate/upon antigenic stimulation, is, compared to natural CD4+regulatory T-cells, further characterised by strong phosphorylation ofPI3K and of AKT. In a further embodiment, not excluding the previousembodiments, said population of cytotoxic CD4+ T-cells is yet furthercharacterised by production, upon antigenic stimulation, of highconcentrations of IFN-gamma with variable concentrations of IL-4, IL-5,IL-10 and TNF-alpha (depending of the cytokine commitment of thecorresponding effector clone), but no or undetectable production ofIL-17 or TGF-beta, all when compared to natural CD4+ regulatory T-cells.More specifically, IL-10 concentrations of activated CD4+ T-cellsaccording to the invention are significantly and drastically reducedcompared to IL-10 concentrations in activated natural CD4+ regulatoryT-cells. In a further embodiment, not excluding the previousembodiments, said population of cytotoxic CD4+ T-cells is yet furthercharacterised by production, upon antigenic stimulation, of highconcentrations of soluble FasL (Fas ligand) compared to natural CD4+regulatory T-cells.

In a further aspect, the current invention encompasses isolatedpopulations of cytotoxic CD4+ T-cells characterised, when compared toCD4+ effector T-cells, by constitutive expression (i.e. independent ofwhether the cytotoxic CD4+ T-cells are at rest or activated) of cellsurface proteins CD25, GITR and intracellular CTLA-4, but no orundetectable expression of CD28 or CD127. In the present inventionconstitutive expression relates to the expression of a protein incytotoxic CD4+ T-cells after a period of rest (i.e. no antigenicstimulation) of about 12 to 15 days. In a further embodiment, notexcluding the previous embodiment, said population of cytotoxic CD4+T-cells is, when compared to CD4+ effector T-cells, furthercharacterised by expression of NKG2D. In a further embodiment, notexcluding the previous embodiments, said population of cytotoxic CD4+T-cells is, upon antigenic stimulation, yet further characterised byproduction of high concentrations of soluble FasL when compared to CD4+effector T-cells. In a further embodiment, not excluding the previousembodiments, said population of cytotoxic CD4+ T-cells is, afterantigenic stimulation, yet further characterised by combined expressionof transcription factors T-bet and GATA3 when compared to CD4+ effectorT-cells. In a further embodiment, not excluding the previousembodiments, said population of cytotoxic CD4+ T-cells is yet furthercharacterised by absence of (detectable) IL-2 transcription whencompared to CD4+ effector T-cells. In a further embodiment, notexcluding the previous embodiments, said population of cytotoxic CD4+T-cells is, when compared to CD4+ effector T-cells, yet furthercharacterised by the capacity to induce apoptosis of APC, afterantigenic stimulation by cognate interaction with peptide presented byMHC class II determinants. In a further embodiment, not excluding theprevious embodiments, said population of cytotoxic CD4+ T-cells is, incomparison with CD4+ effector T-cells, yet further characterised by thecapacity to induce apoptosis of bystander T cells.

In a further aspect, the current invention encompasses isolatedpopulations of cytotoxic CD4+ T-cells characterised, when compared toNK-cells, by expression of the CD4 co-receptor. In a further embodiment,not excluding the previous embodiments, said population of cytotoxicCD4+ T-cells is, when compared to NK-cells, yet further characterised byabsence of CD49b (as detected by binding of antibody DX5). Thesecharacteristics relative to NK-cells are independent of the activationstatus of the cytotoxic CD4+ T-cells and thus are detectable both inresting and activated cells.

In yet a further embodiment of the invention are comprised isolatedpopulations of cytotoxic CD4+ T-cells characterised, when compared toNKT-cells by expression of an alpha-beta T cell receptor with invariantalpha chain and re-arranged beta chain. In a further embodiment, notexcluding the previous embodiments, said population of cytotoxic CD4+T-cells is, when compared to NKT-cells, further characterised by absenceof (detectable) expression of the Valpha14 (mouse) or Valpha24 (human)TCR expression. In a further embodiment, not excluding the previousembodiments, said population of cytotoxic CD4+ T-cells is, in comparisonwith NKT-cells, yet further characterised by lack of CD1d restriction.These characteristics relative to NKT-cells are independent of theactivation status of the cytotoxic CD4+ T-cells and thus are detectableboth in resting and activated cells.

The invention comprises in another aspect isolated populations ofcytotoxic CD4+ T-cells displaying any possible combination of any of thecharacteristics as described above and relative to natural CD4+regulatory T-cells, CD4+ effector T-cells, NK-cells and/or NKT-cells orcharacterised by a combination of all of these characteristics.

The invention relates in another aspect to a method for obtaining orinducing populations of cytotoxic CD4+ T-cells as described aboveaccording to the invention, said methods comprising the steps of:

-   -   (i) providing isolated natural naïve or memory CD4+ T-cells;    -   (ii) contacting said cells with an immunogenic peptide        comprising a T-cell epitope and, adjacent to said T-cell epitope        or separated therefrom by a linker of at most 7 amino acids, a        C-(X)2-[CST] or [CST]-(X)2-C motif; and    -   (iii) expanding said cells in the presence of IL-2.

In a further aspect, the invention encompasses a method of identifying apopulation of cytotoxic CD4+ T-cells, said method comprising the stepsof:

-   -   (i) providing isolated natural CD4+ T-cells such as natural CD4+        regulatory T-cells, CD4+ effector cells, NK-cells or NKT-cells;    -   (ii) providing CD4+ T-cells suspected of being cytotoxic; and    -   (iii) determining that the T-cells provided in (ii) display,        compared to the T-cells provided in (i), the respective        characteristics as described above.

Thus, in one embodiment thereto, said method is identifying cytotoxicCD4+ T-cells by determining in step (iii) the absence of or undetectableexpression of the transcription receptor Foxp3 when compared withexpression of Foxp3 in natural CD4+ regulatory T-cells. Said method mayfurther comprise determining in step (iii) that the T-cells provided in(ii) display, compared to natural CD4+ regulatory T-cells provided in(i), an increased kinase activity of the serine-threonine kinase AKT. Ina further embodiment, not excluding the previous embodiment, said methodis further comprising determining in step (iii) that the T-cellsprovided in (ii) display, compared to natural CD4+ regulatory T-cellsprovided in (i), undetectable production of TGF-beta and undetectable orvery low production of IL-10. In a further embodiment, not excluding theprevious embodiments, said method is further comprising determining instep (iii) that the T-cells provided in (ii) display, compared tonatural CD4+ regulatory T-cells provided in (i), high concentrations ofIFN-gamma production. In a further embodiment, not excluding theprevious embodiments, said method is further comprising determining instep (iii) that the T-cells provided in (ii) display, compared tonatural CD4+ regulatory T-cells provided in (i), production of highconcentrations of soluble FasL.

In a further embodiment said method identifies populations of cytotoxicCD4+ T-cells according to the invention by comparing them with CD4+effector cells. Thus, such methods may comprise determining in step(iii) that the T-cells provided in (ii) display, compared to CD4+effector cells provided in (i), constitutive expression of cell surfaceproteins CD25, GITR and intracellular CTLA-4, but not of CD28 or CD127.In a further embodiment, not excluding the previous embodiments, saidmethod is further comprising determining in step (iii) that the T-cellsprovided in (ii) display, compared to CD4+ effector cells provided in(i), expression of NKG2D on the cell surface. In a further embodiment,not excluding the previous embodiments, said method is furthercomprising determining in step (iii) that the T-cells provided in (ii)display, compared to CD4+ effector cells provided in (i), co-expressionof transcription factors T-bet and GATA3. In a further embodiment, notexcluding the previous embodiments, said method is further comprisingdetermining in step (iii) that the T-cells provided in (ii) display,compared to CD4+ effector cells provided in (i), a absence of IL-2transcription. In a further embodiment, not excluding the previousembodiments, said method is further comprising determining in step (iii)that the T-cells provided in (ii) display, compared to CD4+ effectorcells provided in (i), the capacity to induce apoptosis of APC, afterantigenic stimulation by cognate interaction with peptide presented byMHC class II determinants. In a further embodiment, not excluding theprevious embodiments, said method is further comprising determining instep (iii) that the T-cells provided in (ii) display, compared to CD4+effector cells provided in (i), the capacity to induce apoptosis ofbystander T cells.

In a further embodiment said method identifies populations of cytotoxicCD4+ T-cells according to the invention by comparing them with NK-cells.Thus, such methods may comprise determining in step (iii) that theT-cells provided in (ii) display, compared to NK cells provided in (i),expression of the CD4 co-receptor. In a further embodiment, notexcluding the previous embodiments, said method is further comprisingdetermining in step (iii) that the T-cells provided in (ii) display,compared to NK cells provided in (i), the absence of expression ofCD49b.

In a further embodiment of the invention are included methods foridentifying populations of cytotoxic CD4+ T-cells according to theinvention by comparing them to NKT-cells. Such methods may comprisedetermining in step (iii) that the T-cells provided in (ii) display,compared to NKT-cells provided in (i), expression of an alpha-beta Tcell receptor with rearranged beta chain. In a further embodiment, notexcluding the previous embodiments, said method is further comprisingdetermining in step (iii) that the T-cells provided in (ii) display,compared to NKT-cells provided in (i), absence of expression of theValpha14 (mouse) or Valpha24 (human) TCR expression. In a furtherembodiment, not excluding the previous embodiments, said method isfurther comprising determining in step (iii) that the T-cells providedin (ii) display, compared to the NKT-cells provided in (i), absence ofCD1d restriction.

In the above methods according to the invention it is further possibleto identify CD4+ T-cells suspected to be cytotoxic CD4+ T-cellsaccording to the invention as provided in step (ii) by determining instep (iii) any possible combination of any or all of the characteristicsas described above and relative to natural CD4+ regulatory T-cells, CD4+effector T-cells, NK-cells and/or NKT-cells provided in step (i), saidcombinations also being described above.

FIGURE LEGENDS

FIG. 1. Cytolytic CD4+ T cell clones express markers associated withregulatory T cells. See Example 2 for detailed explanation.

FIG. 2. Cytolytic CD4+ T cell clones co-express transcription factorsT-bet and GATA3 but not Foxp3. See Example 3 for detailed explanation.

FIG. 3. Cytolytic CD4+ T cells are distinct from NK cells. See Example 5for detailed explanation.

FIG. 4. Cytolytic CD4+ T cells are distinct from NKT cells. See Example6 for detailed explanation.

FIG. 5. Cytolytic CD4+ T cells show phosphorylation of AKT by contrastto natural CD4+ regulatory cells. See Example 7 for detailedexplanation.

FIG. 6. Cytolytic CD4+ T cells induce apoptosis of antigen-presentingcells after cognate peptide recognition. See Example 8 for detailedexplanation.

FIG. 7. Cytolytic CD4+ T cells induce apoptosis of bystander T cells.See Example 9 for detailed explanation.

DETAILED DESCRIPTION OF THE INVENTION Definitions

The term “peptide” when used herein refers to a molecule comprising anamino acid sequence of between 2 and 200 amino acids, connected bypeptide bonds, but which can in a particular embodiment comprisenon-amino acid structures (like for example a linking organic compound).Peptides according to the invention can contain any of the conventional20 amino acids or modified versions thereof, or can containnon-naturally occurring amino acids incorporated by chemical peptidesynthesis or by chemical or enzymatic modification.

The term “epitope” when used herein refers to one or several portions(which may define a conformational epitope) of a protein which is/arespecifically recognised and bound by an antibody or a portion thereof(Fab′, Fab2′, etc.) or a receptor presented at the cell surface of a Bor T cell lymphocyte, and which is able, by said binding, to induce animmune response.

The term “antigen” when used herein refers to a structure of amacromolecule comprising one or more hapten(s) and/or comprising one ormore T cell epitopes. Typically, said macromolecule is a protein orpeptide (with or without polysaccharides) or made of proteic compositionand comprises one or more epitopes; said macromolecule can hereinalternatively be referred to as “antigenic protein” or “antigenicpeptide”.

The term “T cell epitope” or “T-cell epitope” in the context of thepresent invention refers to a dominant, sub-dominant or minor T cellepitope, i.e., a part of an antigenic protein that is specificallyrecognised and bound by a receptor at the cell surface of a Tlymphocyte. Whether an epitope is dominant, sub-dominant or minordepends on the immune reaction elicited against the epitope. Dominancedepends on the frequency at which such epitopes are recognised by Tcells and able to activate them, among all the possible T cell epitopesof a protein. In particular, a T cell epitope is an epitope bound by MHCclass I or MHC class II molecules.

The term “CD4+ effector cells” refers to cells belonging to theCD4-positive subset of T-cells whose function is to provide help toother cells, such as, for example B-cells. These effector cells areconventionally reported as Th cells (for T helper cells), with differentsubsets such as Th0, Th1, Th2, and Th17 cells.

The term “immune disorders” or “immune diseases” refers to diseaseswherein a reaction of the immune system is responsible for or sustains amalfunction or non-physiological situation in an organism. Included inimmune disorders include e.g. allergic disorders, autoimmune diseases,alloimmunisation reactions, rejection of viral vectors used in genetherapy/gene vaccination.

In the specification the following acronyms and abbreviations are used:

-   AKT: group of serine/protein kinases comprising AKT 1, AKT2 and AKT3    (also known as Protein Kinase B (PKB))-   CD1d: Thymocyte Antigen CD1D-   CD25: Interleukin 2 Receptor, Alpha Chain (also known as IL2RA,    TCGFR, and TAC antigen)-   CTLA-4: Cytotoxic T-Lymphocyte Antigen 4 (also known as CD152)-   CD49b: Integrin, Alpha-2 (also known as ITGA2, VLAA2)-   FasL: FAS Ligand (also known as TNFSF, APT1LG1, CD95L, CD178)-   Foxp3: Forkhead Box P3 (also known as SCURFIN or JM2)-   GATA3: GATA-Binding Protein 3-   GITR: Glucocorticoid-Induced Tnfr-Related Gene; (also known as AITR)-   IFN: Interferon-   NKG2D: Killer cell lectin-like receptor subfamily K, member 1, (also    known as KLRK1, CD314)-   T-bet: T-Box Expressed In T Cells; (also known as T-BOX 21, TBX21)-   TGF-beta: Transforming Growth Factor Beta-   IL: Interleukin-   NK: natural killer cells-   NKT: natural killer T cells

DETAILED DESCRIPTION

The present invention is based on the characterisation of a subset ofCD4+ T cells having novel characteristics. This new subset of CD4+ Tcells shares characteristics of regulatory T cells, of effector cellsand of NK/NKT cells, but carries features and expresses properties thatclearly distinguish it from regulatory cells, effector cells and NK/NKTcells. Such new CD4+ T cells are called cytolytic (or cytotoxic) CD4+ Tcells (cCD4+ T cells in short). cCD4+ T cells are anergic, long-livingand become activated only when recognising a cognate peptide presentedby antigen-presenting cells (APCs). These cCD4+ T cells are thusstrictly antigen-specific. Furthermore, they can easily be expanded(such as under ex vivo or in vitro conditions) in the presence of IL-2.An additional advantage of the cCD4+ T cells of the invention existstherein that they do not produce suppressive cytokines, which limits therisk of non-specific effects. Clearly, the advantages exhibited by thecCD4+ T cells of the invention make them excellent candidates fortreating immune disorders via adoptive cell transfer.

The cCD4+ T cells of the invention are distinct from natural Tregs byabsence of expression or undetectable expression of the transcriptionrepressor Foxp3, by production of high concentrations of IFN-gamma uponstimulation with variable concentrations of IL-4, IL-5, and TNF-alpha(depending of the cytokine commitment of the corresponding effectorclone), but undetectable, or no IL-17 or TGF-beta (transforming growthfactor beta). In cCD4+ T-cells, IL-10 concentrations are significantlyor drastically reduced or lower compared to natural CD4+ regulatorycells. Furthermore, cCD4+ T-cells according to the invention displaystrong phosphorylation of PI3K and AKT, and production of highconcentrations of soluble FasL, though all these characteristics are notnecessarily present together.

The cCD4+ T cells of the invention are distinct from CD4+ effector Tcells by constitutive expression of cell surface proteins CD25, GITR andintracellular CTLA-4, but no or undetectable expression of CD28 orCD127, by cell expression of NKG2D, by production of high concentrationsof soluble FasL, by co-expression of transcription factors T-bet andGATA3, by absence of IL-2 transcription, by the capacity to induceapoptosis of antigen-presenting cells (APCs), after antigenicstimulation by cognate recognition of peptides presented by MHC class IIdeterminants and by the capacity to induce apoptosis of bystander Tcells, though all these characteristics are not necessarily presenttogether.

The cCD4+ T cells of the invention are distinct from NK cells byexpression of the CD4 co-receptor, by constitutive expression of cellsurface proteins CD25, GITR and intracellular CTLA-4 and by absence of(detectable) CD49b expression. NK cells do not express the CD4co-receptor but do express CD49b

The cCD4+ T cells of the invention are distinct from NKT cells byexpression of an alpha-beta T cell receptor with rearranged beta chain,by absence of Valpha14 (mouse) or Valpha24 (human) TCR expression and bylack of CD1d restriction, though all these characteristics are notnecessarily present together.

Hence, in one aspect, the current invention encompasses isolatedpopulations of cytotoxic CD4+ T-cells (in either resting or activatedstate) characterised, when compared to natural CD4+ regulatory T-cells,by absence of expression or undetectable expression of the transcriptionrepressor Foxp3. In a further embodiment, not excluding the previousembodiment, said population of cytotoxic CD4+ T-cells, in activatedstate/upon antigenic stimulation, is, compared to natural CD4+regulatory T-cells, further characterised by strong phosphorylation ofPI3K and of AKT. In a further embodiment, not excluding the previousembodiments, said population of cytotoxic CD4+ T-cells is yet furthercharacterised by production, upon stimulation, of high concentrations ofIFN-gamma with variable concentrations of IL-4, IL-5, IL-10 andTNF-alpha (depending of the cytokine commitment of the correspondingeffector clone), but no or undetectable production of IL-17 or TGF-beta,all when compared to natural CD4+ regulatory T-cells. More specifically,IL-10 concentrations of activated CD4+ T-cells according to theinvention are significantly and drastically reduced compared to IL-10concentrations in activated natural CD4+ regulatory T-cells. In afurther embodiment, not excluding the previous embodiments, saidpopulation of cytotoxic CD4+ T-cells is yet further characterised byproduction, upon antigenic stimulation, of high concentrations ofsoluble FasL (Fas ligand) compared to natural CD4+ regulatory T-cells.

In a further aspect, the current invention encompasses isolatedpopulations of cytotoxic CD4+ T-cells characterised, when compared toCD4+ effector T-cells, by constitutive expression (i.e. independent ofwhether the cytotoxic CD4+ T-cells are at rest or activated) of cellsurface proteins CD25, GITR and intracellular CTLA-4, but no orundetectable expression of CD28 or CD127. In a further embodiment, notexcluding the previous embodiment, said population of cytotoxic CD4+T-cells is, when compared to CD4+ effector T-cells, furthercharacterised by constitutive expression of NKG2D. In a furtherembodiment, not excluding the previous embodiments, said population ofcytotoxic CD4+ T-cells is, upon antigenic stimulation, yet furthercharacterised by production of high concentrations of soluble FasL whencompared to CD4+ effector T-cells. In a further embodiment, notexcluding the previous embodiments, said population of cytotoxic CD4+T-cells is, after antigenic stimulation, yet further characterised byco-expression of transcription factors T-bet and GATA3 when compared toCD4+ effector T-cells. In a further embodiment, not excluding theprevious embodiments, said population of cytotoxic CD4+ T-cells is yetfurther characterised by absence of (detectable) IL-2 transcription whencompared to CD4+ effector T-cells. In a further embodiment, notexcluding the previous embodiments, said population of cytotoxic CD4+T-cells is, when compared to CD4+ effector T-cells, yet furthercharacterised by the capacity to induce apoptosis of APC, afterantigenic stimulation by cognate interaction with peptide presented byMHC class II determinants. In a further embodiment, not excluding theprevious embodiments, said population of cytotoxic CD4+ T-cells is, incomparison with CD4+ effector T-cells, yet further characterised by thecapacity to induce apoptosis of bystander T cells.

In a further aspect, the current invention encompasses isolatedpopulations of cytotoxic CD4+ T-cells characterised, when compared toNK-cells, by expression of the CD4 co-receptor. In a further embodiment,not excluding the previous embodiments, said population of cytotoxicCD4+ T-cells is, when compared to NK-cells, yet further characterised byabsence of CD49b expression. These characteristics relative to NK-cellsare independent of the activation status of the cytotoxic CD4+ T-cellsand thus are detectable both in resting and activated cells.

In yet a further embodiment of the invention are comprised isolatedpopulations of cytotoxic CD4+ T-cells characterised, when compared toNKT-cells by expression of an alpha-beta T cell receptor withre-arranged beta chain. In a further embodiment, not excluding theprevious embodiments, said population of cytotoxic CD4+ T-cells is, whencompared to NKT-cells, further characterised by absence of (detectable)expression of the Valpha14 (mouse) or Valpha24 (human) TCR expression.In a further embodiment, not excluding the previous embodiments, saidpopulation of cytotoxic CD4+ T-cells is, in comparison with NKT-cells,yet further characterised by lack of CD1d restriction. Thesecharacteristics relative to NKT-cells are independent of the activationstatus of the cytotoxic CD4+ T-cells and thus are detectable both inresting and activated cells.

The invention comprises in another aspect isolated populations ofcytotoxic CD4+ T-cells displaying any possible combination of any of thecharacteristics as described above and relative to natural CD4+regulatory T-cells, CD4+ effector T-cells, NK-cells and/or NKT-cells orcharacterised by a combination of all of these characteristics.

The CD4+ T cells of the invention can be elicited from both naïve CD4+ Tcells as well as from memory CD4+ T cells, more particularly byincubation with T-cell epitopes modified by attachment of a consensusmotif sequence with thioreductase activity ([CST]XX[CST]-motif, wherein[CST] is an amino acid selected from cysteine, serine and threonine, andX can be any amino acid except proline). After elicitation, they can beexpanded in a suitable culture medium comprising IL-2.

The invention relates in another aspect to a method for obtaining orinducing populations of cytotoxic CD4+ T-cells as described aboveaccording to the invention, said methods comprising the steps of:

-   -   (i) providing isolated natural naïve or memory CD4+ T-cells;    -   (ii) contacting said cells with an immunogenic peptide        comprising a T-cell epitope and, adjacent to said T-cell epitope        or separated therefrom by a linker of at most 7 amino acids, a        C-(X)2-[CST] or [CST]-(X)2-C motif; and    -   (iii) expanding said cells in the presence of IL-2.

In this method the cytotoxic CD4+ T-cells can be obtained or induced invivo or ex vivo. When in vivo, steps (i) and (iii) in the above methodare redundant as the contacting of the cells with the T-cell epitope asdescribed in (ii) is occurring by administering the T-cell epitope tothe subject in need thereof, and the resulting cytotoxic CD4+ T-cellswill expand in the subject's body.

The invention relates in another aspect to a method for obtaining orinducing populations of cytotoxic CD4+ T-cells as described aboveaccording to the invention, said methods comprising the steps of:

-   -   (i) administering to a subject in need thereof an immunogenic        peptide comprising a T-cell epitope and, adjacent to said T-cell        epitope or separated therefrom by a linker of at most 7 amino        acids, a C-(X)2-[CST] or [CST]-(X)2-C motif, thereby inducing        cytotoxic CD4+ T-cells; and    -   (ii) isolating or obtaining the cytotoxic CD4+ T-cells induced        in (i).

Alternatively, the cCD4+ T cells according to the invention may beobtained by incubation in the presence of APCs presenting theabove-mentioned immunogenic peptide after transduction or transfectionof the APCs with a genetic construct capable of driving expression ofsuch immunogenic peptide. Such APCs may in fact themselves beadministered to a subject in need to trigger in vivo in said subject theinduction of the beneficial subset of cCD4+ T cells. In anotheralternative method, the cCD4+ T cells can be generated in vivo, i.e. bythe administration of the above-mentioned immunogenic peptide to asubject, and collection of the cCD4+ T cells generated in vivo.Accordingly, the present invention further relates to the generation ofthe cCD4+ T cells of the invention both in vivo and in vitro (ex vivo)using the immunogenic peptides or APCs presenting such immunogenicpeptides.

Subjects suffering from, or having an immune disorder, or whom arediagnosed to be predestined for developing an immune disorder can betreated by administering (a sufficient or effective amount of) the cCD4+T-cells according to the invention wherein said cCD4+ T-cells arespecific to a T-cell epitope relevant to the immune disorder to betreated or prevented. Prophylactic administration, treatment orprevention of immune disorders would be desirable in subjectspredestined for developing an immune disorder. Such predestination maybe diagnosed e.g. by a positive diagnosis of a genetic defect known topredestine a subject to develop an immune disorder or known to increasethe likelihood of developing an immune disorder. Alternatively,inheritable immune disorders which have manifested themselves in one ormore of the ancestors or within the family of a subject may increase thechance that/may be predestining said subject to develop the immunedisorder, such subjects may therefore also be eligible for prophylactictreatment with the cCD4+ T-cells according to the invention.

The cCD4+ T cells obtainable by the above methods are of particularinterest for use in the manufacture of a medicament for(prophylactically) preventing, suppressing or treating an immunedisorder in a mammal. Both the use of allogeneic and autogeneic cCD4+T-cells is envisaged. Any method comprising the administration of saidcCD4+ T cells to a subject in need is known as adoptive cell therapy. Asmentioned before, the cCD4+ T-cells to be included in the medicamentwould need to be “educated”, i.e. would need to be specific, for aT-cell epitope of an antigen known to be relevant in the to be treatedimmune disorder.

The above-mentioned immunogenic peptides in general comprise (i) atleast one T-cell epitope of an antigen of choice with a potential totrigger an immune reaction, which is coupled to (ii) an organic compoundhaving a reducing activity, such as a thioreductase sequence motif. Theantigen of choice will vary along with (and be determined by) the immunedisorder to be prevented or suppressed. The T-cell epitope and theorganic compound are optionally separated by a linker sequence. Infurther optional embodiments the immunogenic peptide additionallycomprises an endosome targeting sequence (e.g. late endosomal targetingsequence) and/or additional “flanking” sequences. The immunogenicpeptides can be schematically represented as A-L-B or B-L-A, wherein Arepresents a T-cell epitope of an antigen (self or non-self) with apotential to trigger an immune reaction, L represents a linker and Brepresents an organic compound having a reducing activity. The reducingactivity of an organic compound can be assayed for its ability to reducea sulfhydryl group such as in the insulin solubility assay known in theart, wherein the solubility of insulin is altered upon reduction, orwith a fluorescence-labelled insulin. The reducing organic compound maybe coupled at the amino-terminus side of the T-cell epitope or at thecarboxy-terminus of the T-cell epitope.

Generally the organic compound with reducing activity is a peptidesequence. Peptide fragments with reducing activity are encountered inthioreductases which are small disulfide reducing enzymes includingglutaredoxins, nucleoredoxins, thioredoxins and other thiol/disulfideoxydoreductases They exert reducing activity for disulfide bonds onproteins (such as enzymes) through redox active cysteines withinconserved active domain consensus sequences: C-X(2)-C, C-X(2)-S,C-X(2)-T, S-X(2)-C, T-X(2)-C (Fomenko et al. (2003) Biochemistry 42,11214-11225), in which X stands for any amino acid. Such domains arealso found in larger proteins such as protein disulfide isomerase (PDI)and phosphoinositide-specific phospholipase C. In particular, theimmunogenic peptides comprise as redox motif the thioreductase sequencemotif [CST]-X(2)-[CST], in a further embodiment thereto, said[CST]-X(2)-[CST] motif is positioned N-terminally of the T-cell epitope.More specifically, in said redox motif at least one of the [CST]positions is occupied by a Cys; thus the motif is either [C]-X(2)-[CST]or [CST]-X(2)-[C]. In the present application such a tetrapeptide willbe referred to as “the motif” or “redox motif”. More in particular, theimmunogenic peptides can contain the sequence motif [C]-X(2)-[CS] or[CS]-X(2)-[C]. Even more particularly, the immunogenic peptides containthe sequence motif C-X(2)-S, S-X(2)-C or C-X(2)-C.

The above immunogenic peptides can be made by chemical synthesis, whichallows the incorporation of non-natural amino acids. Accordingly, in theredox motif the C representing cysteine can be replaced by another aminoacids with a thiol group such as mercaptovaline, homocysteine or othernatural or non-natural amino acids with a thiol function. In order tohave reducing activity, the cysteines present in the motif should notoccur as part of a cystine disulfide bridge. Nevertheless, the motif maycomprise modified cysteines such as methylated cysteine, which isconverted into cysteine with free thiol groups in vivo. The amino acid Xin the [CST]-X(2)-[CST] motif of particular embodiments of the reducingcompounds of the invention can be any natural amino acid, including S,C, or T or can be a non-natural amino acid. In particular, X can be anamino acid with a small side chain such as Gly, Ala, Ser or Thr. Moreparticularly, X is not an amino acid with a bulky side chain such asTyr; or at least one X in the [CST]-X(2)-[CST] motif can be His or Pro.

The motif in the above immunogenic peptides is placed either immediatelyadjacent to the epitope sequence within the peptide, or is separatedfrom the T cell epitope by a linker. More particularly, the linkercomprises an amino acid sequence of 7 amino acids or less. Mostparticularly, the linker comprises 1, 2, 3, or 4 amino acids.Alternatively, a linker may comprise 6, 8 or 10 amino acids. Typicalamino acids used in linkers are serine and threonine. Example ofpeptides with linkers in accordance with the present invention areCXXC-G-epitope (SEQ ID NO:17), CXXC-GG-epitope (SEQ ID NO:18),CXXC-SSS-epitope (SEQ ID NO:19), CXXC-SGSG-epitope (SEQ ID NO:20) andthe like.

The immunogenic peptides can comprise additional short amino acidsequences N or C-terminally of the (artificial) sequence comprising theT cell epitope and the reducing compound (motif). Such an amino acidsequence is generally referred to herein as a ‘flanking sequence’. Aflanking sequence can be positioned N- and/or C-terminally of the redoxmotif and/or of the T-cell epitope in the immunogenic peptide. When theimmunogenic peptide comprises an endosomal targeting sequence, aflanking sequence can be present between the epitope and an endosomaltargeting sequence and/or between the reducing compound (e.g. motif) andan endosomal targeting sequence. More particularly a flanking sequenceis a sequence of up to 10 amino acids, or of in between 1 and 7 aminoacids, such as a sequence of 2 amino acids.

In particular embodiments of the invention, the redox motif in theimmunogenic peptide is located N-terminally from the epitope.

As detailed above, the immunogenic peptides comprise a reducing motif asdescribed herein linked to a T cell epitope sequence. In particularcases, the T-cell epitopes are derived from proteins which do notcomprise within their native natural sequence an amino acid sequencewith redox properties within a sequence of 11 amino acids N- orC-terminally adjacent to the T-cell epitope of interest.

In particular embodiments, the T-cell epitope is derived from anallergen or an auto-antigen.

Allergens that can be used for selection of T-cell epitopes aretypically allergens such as:

-   -   food allergens present for example in peanuts, fish e.g.        codfish, egg white, crustacea e.g. shrimp, milk e.g. cow's milk,        wheat, cereals, fruits of the Rosacea family, vegetables of the        Liliacea, Cruciferae, Solanaceae and Umbelliferae families, tree        nuts, sesame, peanut, soybean and other legume family allergens,        spices, melon, avocado, mango, fig, banana;    -   house dust mites allergens obtained from Dermatophagoides spp or        D.

pteronyssinus, D. farinae and D. microceras, Euroglyphus maynei orBlomia sp.,

-   -   allergens from insects present in cockroach or Hymenoptera,    -   allergens from pollen, especially pollens of tree, grass and        weed,    -   allergens from animals, especially in cat, dog, horse and        rodent,    -   allergens from fungi, especially from Aspergillus, Alternaria or        Cladosporium, and    -   occupational allergens present in products such as latex,        amylase, etc.

Auto-antigens that can be used for selection of T-cell epitopes aretypically antigens such as:

-   -   thyroglobulin, thyroid peroxidise or TSH receptor (thyroid        autoimmune diseases);    -   insulin (proinsulin), glutamic acid decarboxylase (GAD),        tyrosine phosphatise IA-2, heat-shock protein HSP65,        islet-specific glucose-6-phosphate catalytic subunit related        protein (IGRP) (type 1 diabetes);    -   21-OH hydroxylase (adrenalitis);    -   17-alpha hydroxylase, histidine decarboxylase, Trp hydroxylase,        Tyr hydroxylase (polyendocrine syndromes);    -   H+/K+ ATPase intrinsic factor (gastritis & pernicious anemia);    -   myelin oligodendrocyte glycoprotein (MOG), myelin basic protein        (MBP), proteolipid protein (PLP) (multiple sclerosis);    -   acetyl-choline receptor (myasthenia gravis);    -   retinol-binding protein (RBP) (ocular diseases);    -   type II (rheumatoid arthritis), type II and type IX collagen        (inner ear diseases);    -   tissue transglutaminase (celiac disease);    -   pANCA histone H1 protein (inflammatory bowel diseases);    -   heat-shock protein HSP60 (atherosclerosis);    -   angiotensin receptor (arterial hypertension and pre-eclampsia)    -   nitrated alpha-synuclein (Parkinson disease)

Other antigens that can be used for selection of T-cell epitopes includealloantigenic proteins derived from (present in/shed from) allograftedcells or organs, soluble alloproteins (such as in administered inreplacement therapy), viral vector proteins as used in gene therapy/genevaccination, antigens derived from intracellular pathogens, and antigensderived from tumours or tumour cells.

In a further aspect, the invention encompasses a method of identifying apopulation of cytotoxic CD4+ T-cells, said method comprising the stepsof:

-   -   (i) providing isolated natural CD4+ T-cells such as natural CD4+        regulatory T-cells, CD4+ effector cells, NK-cells or NKT-cells;    -   (ii) providing CD4+ T-cells suspected of being cytotoxic; and    -   (iii) determining that the T-cells provided in (ii) display,        compared to the T-cells provided in (i), the respective        characteristics as described above.

In particular, the cells to be provided in step (ii) are obtainable byor may be induced or obtained by the above-described method of theinvention. The cells provided in (i) are of a source such that they arenot comprising cytotoxic CD4+ T-cells according to the invention.Depending on the characteristic to be determined in step (iii), thecells provided in steps (i) and/or (ii) may need to be activated by acognate T-cell epitope; said need is derivable from the characteristicsof the CD4+ T-cells of the invention as described above. Theabove-mentioned method of identifying a population of cytotoxic CD4+T-cells of the invention can thus be formulated in a more extensive wayas follows:

-   -   (i) providing isolated natural CD4+ T-cells such as natural CD4+        regulatory T-cells, CD4+ effector cells, NK-cells or NKT-cells        and, optionally, or when required, activating these cells;    -   (ii) providing CD4+ T-cells suspected of being cytotoxic, said        cells being inducible or obtainable as described above, and,        optionally, or when required, activating these cells; and    -   (iii) determining that the T-cells provided in (ii) display,        compared to the T-cells provided in (i), the respective        characteristics as described above.

Thus, in one embodiment thereto, said method is identifying cytotoxicCD4+ T-cells by determining in step (iii) the absence of or undetectableexpression of the transcription receptor Foxp3 when compared withexpression of Foxp3 in natural CD4+ regulatory T-cells. Said method mayfurther comprise determining in step (iii) that the T-cells provided in(ii) display, compared to natural CD4+ regulatory T-cells provided in(i), an increased kinase activity of the serine-threonine kinase AKT. Ina further embodiment, not excluding the previous embodiment, said methodis further comprising determining in step (iii) that the T-cellsprovided in (ii) display, compared to natural CD4+ regulatory T-cellsprovided in (i), undetectable production of TGF-beta and undetectable orvery low production of IL-10. In a further embodiment, not excluding theprevious embodiments, said method is further comprising determining instep (iii) that the T-cells provided in (ii) display, compared tonatural CD4+ regulatory T-cells provided in (i), high concentrations ofIFN-gamma production. In a further embodiment, not excluding theprevious embodiments, said method is further comprising determining instep (iii) that the T-cells provided in (ii) display, compared tonatural CD4+ regulatory T-cells provided in (i), production of highconcentrations of soluble FasL.

In a further embodiment, said method identifies populations of cytotoxicCD4+ T-cells according to the invention by comparing them with CD4+effector cells. Thus, such methods may comprise determining in step(iii) that the T-cells provided in (ii) display, compared to CD4+effector cells provided in (i), constitutive expression of Cell surfaceproteins CD25, GITR and intracellular CTLA-4, but not of CD28 or CD127.In a further embodiment, not excluding the previous embodiments, saidmethod is further comprising determining in step (iii) that the T-cellsprovided in (ii) display, compared to CD4+ effector cells provided in(i), expression of NKG2D on the cell surface. In a further embodiment,not excluding the previous embodiments, said method is furthercomprising determining in step (iii) that the T-cells provided in (ii)display, compared to CD4+ effector cells provided in (i), combinedexpression of transcription factors T-bet and GATA3. In a furtherembodiment, not excluding the previous embodiments, said method isfurther comprising determining in step (iii) that the T-cells providedin (ii) display, compared to CD4+ effector cells provided in (i), aabsence of IL-2 transcription. In a further embodiment, not excludingthe previous embodiments, said method is further comprising determiningin step (iii) that the T-cells provided in (ii) display, compared toCD4+ effector cells provided in (i), the capacity to induce apoptosis ofAPC, after antigenic stimulation by cognate interaction with peptidepresented by MHC class II determinants. In a further embodiment, notexcluding the previous embodiments, said method is further comprisingdetermining in step (iii) that the T-cells provided in (ii) display,compared to CD4+ effector cells provided in (i), the capacity to induceapoptosis of bystander T cells.

In a further embodiment said method identifies populations of cytotoxicCD4+ T-cells according to the invention by comparing them with NK-cells.Thus, such methods may comprise determining in step (iii) that theT-cells provided in (ii) display, compared to NK cells provided in (i),expression of the CD4 co-receptor. In a further embodiment, notexcluding the previous embodiments, said method is further comprisingdetermining in step (iii) that the T-cells provided in (ii) display,compared to NK cells provided in (i), the absence of expression ofCD49b.

In a further embodiment of the invention are included methods foridentifying populations of cytotoxic CD4+ T-cells according to theinvention by comparing them to NKT-cells. Such methods may comprisedetermining in step (iii) that the T-cells provided in (ii) display,compared to NKT-cells provided in (i), expression of an alpha-beta Tcell receptor with rearranged beta chain. In a further embodiment, notexcluding the previous embodiments, said method is further comprisingdetermining in step (iii) that the T-cells provided in (ii) display,compared to NKT-cells provided in (i), absence of expression of theValpha14 (mouse) or Valpha24 (human) TCR expression. In a furtherembodiment, not excluding the previous embodiments, said method isfurther comprising determining in step (iii) that the T-cells providedin (ii) display, compared to the NKT-cells provided in (i), absence ofCD1d restriction.

In the above methods according to the invention it is further possibleto identify CD4+ T-cells suspected to be cytotoxic CD4+ T-cellsaccording to the invention as provided in step (ii) by determining instep (iii) any possible combination of any or all of the characteristicsas described above and relative to natural CD4+ regulatory T-cells, CD4+effector T-cells, NK-cells and/or NKT-cells provided in step (i), saidcombinations also being described above.

Accordingly, the invention provides different markers and functionalproperties, which can be used alone or in combination to identify and/orselect and/or to use in the quality control of cCD4+ T cells. Inparticular embodiments the methods comprise a comparison with naturalregulatory T-cells, CD4+ effector cells and NK/NKT cells. However, it isenvisaged that in particular embodiments, determining the concentrationof the markers mentioned above as such is sufficient to identify thecells (based upon the known expression concentrations or functionalproperties in natural regulatory T-cells, CD4+ effector cells and NK/NKTcells). Accordingly determining the increased activity or expression ofa marker can optionally also involve determining ‘high’ concentrationsof expression of such marker.

Generally, an enhanced activity of a kinase can be caused by anincreased expression of that kinase or byphosphorylation/dephosphorylation of the kinase itself which increasesits enzymatic activity. The activity of a kinase is determined bymeasuring directly or indirectly the amount of phosphate that isincorporated in a natural or model substrate (e.g. synthetic peptide).The activity of a kinase often depends on the phosphorylation of thatsame kinase. Accordingly, the degree of phosphorylation of a kinase canbe indicative for its activity as is the case for AKT kinase. In theabove the extent of kinase activity of the serine-threonine kinase AKTand of PI3K can be estimated via Western blotting using an antibodyspecific to the phosphorylated AKT or PI3K, respectively. Thephosphorylation can be qualified by densitometric scanning of theWestern blot. Other quantitative methods comprise methods whereinWestern blots are quantified with chemoluminescence techniques (e.g.phosphorimaging) Alternatively phosphorylation can be determinedquantitatively by measuring the incorporation of radioactive phosphateinto a substrate. Expression of the transcription repressor Foxp3, oftranscription activators T-bet and GATA3 and IL-2 expression can beestimated via Northern or RNA blotting using a labelled probe specificto the respective transcript. Expression levels can subsequently bequalified by densitometric scanning of the Northern blot. Expressionlevels of certain markers can alternatively be determined at the mRNAlevel by reverse transcriptase PCR methods. Undetectable expression asdetermined by RT-PCR refers to experiments wherein no signal is detectedafter 35 cycles of amplification.

Expression of surface markers can be evaluated using specific antibodiesand a fluorescence-activated cell sorter (Facs). Facs analysis allows todetermine the relative amount of cells which express a certain marker ora combination of markers. In this context, undetectable expression of amarker (for example of Foxp3), relates to a population of cells whereinless than 1%, less than 0.5% or even less than 0.1% of the cells expresssaid marker or combination of markers.

Facs analysis is in the present invention also used to determine whethertwo or more markers are co-expressed. In this context two proteins areconsidered as co-expressed when at least 70, 80, 90, 95 or 99% of thecells in a cell population stain positive for said two or more markersin a Facs analysis.

Production of cytokines such as IL-10 and TGF-beta, IFN-gamma, IL-4,IL-5, IL-17 and IL-13, and of soluble FasL were determined in thisinvention via ELISA, but can also be determined via an ELISPOT assay.Cytokine concentrations are quantifiable via optical densitydetermination in solution (ELISA) or spots indicating the presence ofcytokines can be counted manually (e.g., with a dissecting microscope)or using an automated reader to capture the microwell images and toanalyse spot number and size (ELISPOT). The production of cytokines asdetermined by ELISA is considered to be “undetectable” when theconcentration is below 50 pg/ml, below 20 pg/ml or even below 10 pg/ml,and may depend from the type of antibody and the supplier). Theproduction of cytokines as determined by ELISA is considered to be “verylow” when the concentration is between 50 to 1000 pg/ml, between 100 to1000 pg/ml, or between 200 to 1000 pg/ml. The production of cytokines asdetermined by ELISA is considered to be “high” when the concentration isabove 1000 pg/ml, 2000 pg/ml, 5000 pg/ml or even above 7500 pg/ml.

The production of transmembrane proteins (such as FasL) as determined byELISA is considered to be “high” when the concentration is above 50pg/ml, 75 pg/ml, 100 pg/ml or even above 150 pg/ml.

Generally, concentration measurements refer to conditions wherein theprotein production of about 100,000 cells is assayed in a volume of 200μl.

Induction of apoptosis in APCs or bystander T cells can be measured byevaluating the binding of annexin V to phosphatidylserine exposed as theresult of apoptosis.

The increase of kinase activity of the serine-threonine kinase AKT inthe cytolytic or cytotoxic CD4+ regulatory T-cells of the invention isabout 2-fold compared to natural CD4+ regulatory T-cells, or can be upto 3-, 4-, 5-, 5.5-, 6-, 7-, 8-, 9- or 10-fold, and can be determined bymethods known in the art as explained above.

The present invention will now be illustrated by means of the followingexamples, which are provided without any limiting intention.Furthermore, all references described herein are explicitly includedherein by reference.

EXAMPLES Example 1 List of Peptides Used in the Examples

SEQ ID NO:1, CHGSEPCIIHRGKPF (referred to in Figures as Der p2),corresponding to amino acid sequence 21 to 35 of allergen Der p 2 andcontaining a T cell epitope and a natural thioreductase sequence(underlined).

SEQ ID NO:2, CGPCGGYRSPFSRVVHLYRNGK (referred to in Figures as MOG+),corresponding to amino acid sequence 40-55 derived from the myelinoligodendrocytic glycoprotein (MOG) and modified by addition of athioreductase motif (underlined) separated from the first MHC class IIanchoring residue by a Gly-Gly sequence.

SEQ ID NO:3, CGPCGGYVPFHIQVP (referred to in Figures as LP HAdV5),corresponding to amino acid sequence 555-563 from Late Protein 2 (hexonprotein family) derived from human adenovirus 5 (HAdV-5) and modified byaddition of a thioreductase motif (underlined) separated from the firstMHC class II anchoring residue by a Gly-Gly sequence.

SEQ ID NO:4, CGHCGGAAHAEINEAGR (referred to in Figures as OVA+),corresponding to amino acid sequence 330-340 derived from chickenovalbumin and modified by addition of a thioreductase motif (underlined)separated from the first MHC class II anchoring residue by a Gly-Glysequence.

SEQ ID NO:5, CHGCGGEPCIIHRGKPF (referred to in Figures as Der p2+),corresponding to amino acid sequence 25 to 35 of allergen Der p 2 andmodified by addition of a thioreductase motif (underlined) separatedfrom the first MHC class II anchoring residue by a Gly-Gly sequence.

SEQ ID NO:6, YRSPFSRVVHLYRNGK (referred to in Figures as MOG-),corresponding to amino acid sequence 40-55 derived from the myelinoligodendrocytic glycoprotein (MOG).

SEQ ID NO:7, IIARYIRLHPTHYSIRST (referred to in Figures as fVIII-),corresponding to amino acid sequence 2144-2161 derived from the Cldomain of human Factor VIII.

SEQ ID NO:8, CGFSSNYCQIYPPNANKIR (referred to in Figures as Der p1 +),corresponding to amino acid sequence 114 to 128 of allergen Der p 1 andmodified by addition of a thioreductase motif (underlined) to theamino-terminal 2 0 part of the first MHC class II anchoring residue.

SEQ ID NO:9, NACHYMKCPLVKGQQ (referred to in Figures as Der p2*-),corresponding to amino acid sequence 71 to 85 of allergen Der p 2.

SEQ ID NO:10, CHGAEPCIIHRGKPF (referred to in Figures as Der p2mut),corresponding to peptide of SEQ ID1 containing a single S to A mutation(underlined) that abolishes the thioreductase activity of peptide.

SEQ ID NO:11, TYLRLVKIN (referred to in Figures as gD HSV1-),corresponding to amino acid sequence 188 to 196 derived fromglycoprotein D of human herpesvirus 1.

SEQ ID NO:12, CGHCTYLRLVKIN (referred to in Figures as gD HSV1+),corresponding to amino acid sequence 188 to 196 derived fromglycoprotein D of human herpesvirus 1 and modified by addition of athioreductase motif (underlined) to the amino-terminal part of the firstMHC class II anchoring residue.

SEQ ID NO:13, SNYCQIYPPNANKIR (referred to in Figures as Der p1-),corresponding to amino acid sequence 114 to 128 of allergen Der p 1.

SEQ ID NO:14, ISQAVHAAHAEINEAGR (referred to in Figures as OVA−)corresponding to amino acid sequence 324-340 derived from chickenovalbumin.

Example 2 Cytolytic CD4+ T cell Clones Express Markers Associated withRegulatory T Cells

The phenotype of natural regulatory T cells is characterised by highexpression of CD25 at rest, together with high expression ofintracellular CTLA-4 and surface GITR (Glucocorticoid-Induced TNFreceptor), which distinguish regulatory T cells form effector cells.

Peptides derived from 4 distinct antigens were used: an allergen, anautoantigen and a virus-derived surface antigen and a common antigen.

CD4+ T cells were obtained from the spleen of BALB/c mice immunised withpeptide p21-35 (SEQ ID NO:1, CHGSEPCIIHRGKPF), followed by purificationby magnetic beads sorting. A T cell clone was obtained by in vitrostimulation with peptide-loaded APCs (loaded with peptide of SEQ IDNO:1). The clonal cells were analysed on day 15 after stimulation byfluorescence-activated cell sorting (Facs) using a FACSCalibur© flowcytometer. CD4+ T cells were stained with an antibody recognising CD25,then permeabilised with saponin before incubation with an antibodyspecific for CTLA-4. Data show strong positivity for both CD25 andCTLA-4 (FIG. 1A). The T cell clone was also tested for expression ofGITR and CD28, showing a strong positivity for GITR (FIG. 1D), butabsence of CD28 (FIG. 1F). A CD4+ T cell clone obtained after mouseimmunisation with peptide of SEQ ID NO:2 (CGPCGGYRSPFSRVVHLYRNGK) wastested for the expression of CD25 (FIG. 1B) and CD28 (FIG. 1G), showingstrong CD25 expression but absence of CD28. Further, CD25 expression(FIG. 10) or absence of CD28 expression (FIG. 1H) was shown for a cloneobtained after immunisation with peptide of SEQ ID NO:3(CGPCGGYVPFHIQVP). Expression of surface GITR, an hallmark of the 3clones shown above was also observed with a CD4 T cell clone specificfor peptide of SEQ ID NO:4 (CGHCGGAAHAEINEAGR), FIG. 1 E.

Example 3 Cytolytic CD4+ T Cell Clones Co-Express Transcription FactorsT-Bet and GATA3 but Not Foxp3

T-bet is considered as a marker for Th1 cells and GATA3 as a marker forTh2 cells. In helper cells, expression of T-bet excludes expression ofGATA3 and vice-versa.

A T cell clone was obtained as described in Example 2 with a peptide ofSEQ ID NO:1. After antigenic stimulation, cells were fixed andpermeabilised before intracellular staining with specific antibodies toeither Foxp3, T-bet or GATA3 and analysed by Facs as described inExample 2. Cells are shown to be positive for both T-bet and GATA3 butnot for Foxp3 (FIG. 2A). Dual staining with T-bet and GATA-3 of a T cellclone obtained by immunisation with peptide of SEQ ID NO:4 (FIG. 2B,upper panel) of by peptide of SEQ ID NO:2 (FIG. 2C, lower panel) is alsoshown.

Example 4 Cytolytic CD4+ T Cell Clones Produce Soluble FasL andIFN-Gamma

The profile of cytokines produced by effector cells characterises thesubset to which cells belong. Th1 cells produce IL-2, IFN-gamma andTNF-alpha, Th2 cells produce IL-4, IL-5, IL-13 and IL-10, and Th17 cellsproduce IL-17 and IL-6.

Two distinct T cell clones were obtained from 2 mice immunised with apeptide containing a thioreductase motif (SEQ ID NO:5,CHGCGGEPCIIHRGKPF).

In addition, T cell lines were obtained from mice immunised with a Tcell epitope in natural sequence (SEQ ID NO:6, YRSPFSRVVHLYRNGK) derivedfrom the myelin oligodendrocytic glycoprotein (MOG) and were stimulatedin vitro in the presence of the same T cell epitope modified by additionof a thioreductase motif (underlined) separated from the first MHC classII anchoring residue by a Gly-Gly sequence (SEQ ID NO:2,CGPCGGYRSPFSRVVHLYRNGK).

The two CD4 T cell clones specific for SEQ ID NO:5 and a CD4 T cell linespecific to SEQ ID NO:6 were stimulated with peptides for 48 h and thesupernatants were assessed for the presence of cytokines and of FasLusing

ELISAs with specific antibodies. Table 1 shows that the two CD4 T cellclones obtained from immunisation with peptide of SEQ ID NO:5 and theCD4 T cell clone obtained with the natural sequence SEQ ID NO:6, whenstimulated in vitro with either peptide of SEQ ID NO:5 for the first twoclones or peptide of SEQ ID NO:2 for the third clone, significantamounts of soluble FAS-L was detected in the supernatants. Bycomparison, the T cell clone obtained by immunization with peptide ofSEQ ID NO:6 was stimulated in vitro with the same peptide, no FAS-L wasdetected. An additional control is shown, made from a T cell clonestimulated by peptide of SEQ ID NO:7 (IIARYIRLHPTHYSIRST, a T cellepitope derived from human Factor VIII), which does not contain athioreductase motif.

TABLE 1 T cell Specificity sFAS-L (pg/ml) cCD4 T (R3TB7) to SEQ ID 115.1NO: 5 cCD4 T (22N) to SEQ ID NO: 5 176.1 cCD4 T to SEQ ID NO: 2  50.8CD4 T to SEQ ID NO: 6 ND CD4 T (p352a) to SEQ ID NO: 7 ND

A consistent finding with all clones obtained by immunisation withpeptides containing a thioreductase motif, or effector CD4 T cellsstimulated in vitro with peptides containing a thioreductase motif, wasthe sustained production of IFN-gamma, while IL-2, TGF-beta and IL-17were not detected (ND). Low concentrations of IL-4, IL-5 and IL-10 couldbe detected, which correlated with the cytokine profile of thecorresponding effector cell (Table 2).

TABLE 2 TGF-β IL-17 IFN-γ IL-5 IL-4 IL-10 IL-2 cCD4 T (R3TB7) to ND ND4151 8 ND ND ND SEQ ID NO: 5 cCD4 T (22N) to ND ND 9139 2 ND  102 ND SEQID NO: 5 cCD4 T to ND ND 133 16 ND ND ND SEQ ID NO: 2 CD4 T to ND ND4652 2538  42 5001  7 SEQ ID NO: 6 CD4 T (p352a) to ND ND 131 725 2523847 19 SEQ ID NO: 7 cytokine concentrations are expressed as pg/ml

Example 5 Cytolytic CD4+ T Cells are Distinct from NK Cells

NK cells are characterised by expression of CD49b and NKG2D but not

CD4.

T cell clones were obtained from mice immunised with peptides of SEQ IDNO:5 or of SEQ ID NO:4. Such clones were analysed byfluorescence-activated cell sorting for the expression of CD49b cellmarker on day 14 after in vitro restimulation. Antibodies specific toCD49b (DX5 antibody) were used in the FACS analysis.

The results indicated that the two clones (FIGS. 3A and FIG. 3B,respectively) were uniformly negative for CD49b, thereby distinguishingcytolytic T cell clones from NK cells.

FIGS. 3C and 3D show the expression of NKG2D on cytolytic CD4 cellsobtained from mice immunised with peptide of SEQ ID NO:5 or peptide ofSEQ ID NO:8 (CGFSSNYCQIYPPNANKIR), respectively.

By comparison, expression of NKG2D was evaluated on effector CD4 T cellclones obtained by immunisation by peptide of SEQ ID NO:9(NACHYMKCPLVKGQQ) or of SEQ ID NO:7, containing no thioreductase motif(FIGS. 3E and 3F, respectively) and effector CD4 T cells obtained byimmunisation with a full allergen, Der p2 (FIG. 3G, denoted in FIG. 3Gas Der p2 FL). None of these non-cytolytic effector CD4 T cellsexpressed NKG2D.

Example 6 Cytolytic CD4+ T Cells are Distinct from NKT Cells

NKT cells carry an invariant alpha chain (Valpha14-Jalpha281 in themouse, Valpha24-JQ in man) and variable but not rearranged beta chain atthe TCR level. In addition, NKT cells produce high concentrations ofIL-4, and most NKT cells are restricted by the CD1d molecule. CD1drestriction refers to the fact that the recognition of an antigen loadedby CD1d+ cell (antigen presenting cell) is mediated through therecognition of the antigen by a T cell when such antigen is presented bythe CD1d molecule. In the present example the antigen presenting cell isreplaced by a soluble from of CD1d (BDTM DimerX; Becton Dickinson)

A T cell clone obtained as in Example 5 by stimulation with peptide ofSEQ ID NO:5 was assessed on day 14 after restimulation. Table 2 showsthat such clone (R3TB7) does not produce detectable concentrations ofIL-4, distinguishing this clone from NKT cells. Cells were furtheranalysed by Facs using specific antibodies to the Vbeta8-1 TCR (FIG.4A). In addition, the sequence of the alpha chain of the TCR wasobtained by PCR. The results indicate that the clone expressed arearranged Vbeta chain and a Valpha sequence belonging to the Valpha5subfamily (and not the sequence of the invariant Valpha14-Jalpha281 TCRchain), thereby distinguishing the cytolytic CD4+ T cells (R3TB7) fromNKT cells (FIG. 4B). The T cell clone was further tested for stainingwith peptide of SEQ ID NO:5-loaded CD1d-Ig molecule (BDTM DimerX; BectonDickinson) and analysed by Facs. This experiment shows that either theantigenic peptide is not loaded on soluble CD1d and or that the cCD4+Tcell is not equipped with the appropriate receptor to recognize thepeptide as associated with the CD1d molecule.

The results indicate that the cytolytic CD4+ cells were not restrictedby CD1 d molecule, further distinguishing them from CD4+ NKT cells (FIG.4C). Data is representative of different clones with distinct antigenspecificity.

Example 7 Cytolytic CD4+ T Cells Show Phosphorylation of AKT by Contrastto Natural CD4+ Regulatory Cells

In CD4 T lymphocytes the phosphorylation of Ser473 of serine-threoninekinase AKT is indicative of its activity. To show that AKT kinaseactivity was present and/or increased when CD4+ T cells were incubatedwith peptides containing a thioreductase motif, we made use of peptideof SEQ ID NO:5 (containing such a motif) and peptide of SEQ ID NO:10(CHGAEPCIIHRGKPF, containing a single S to A mutation (underlined) thatabolishes the thioreductase activity of peptide of SEQ ID NO:1).

The cytolytic CD4+ T cell clone R3TB7 was obtained by immunisation withpeptide of SEQ ID NO:1, followed by cloning, and amplification in thepresence of dendritic cells presenting the same peptide. The R3TB7 CD4+T cell clone was incubated for 30 minutes with antigen-presenting cells(dendritic cells) without peptide (FIG. 5, lane 1), with APC preloadedwith redox-inactive peptide of SEQ ID NO:10 (FIG. 5, lane 2), or withAPC preloaded with a redox active peptide of SEQ ID NO:5 (FIG. 5, lane3). Cells were then lysed and an aliquot was run on SDS-PAGE, theproteins were then transferred to a PVDF membrane and probed for thephosphorylated form of AKT (Ser473) using a specific antibody. A controlcontaining no T cells was also included (lane 4). The resultingphosphorylation of the serine-threonine kinase AKT in CD4+ T-cellsincubated with redox-active peptide of SEQ ID NO:5 was 5,5-fold higheras compared to AKT in the same CD4+ T-cells incubated without unloadedAPCs (FIG. 5, lane 1, mimicking natural CD4+ regulatory T-cells), and2-fold higher compared to AKT in the same CD4+ T-cells incubated withthe redox-inactive peptide of SEQ ID NO:10.

Thus, a CD4+ T cell clone obtained from animals immunised with athioreductase containing peptide shows strong kinase activity of AKTwhen incubated in vitro with a peptide containing a thioreductaseactivity (peptide of SEQ ID NO:5), yet maintains AKT kinase activitywhen incubated with a peptide from which the thioreductase activity hasbeen removed by mutation (peptide of SEQ ID NO:10), illustrating thestable commitment of such T cell clone under in vitro stimulationconditions. These results as illustrated were obtained after a singleincubation of cells with peptides. This unexpected observation contrastswith the results disclosed by Crellin et al. (2007) (Blood 109,2014-2022) showing a decreased kinase activity of AKT to be associatedwith natural CD4+ regulatory T-cells, and emphasises the differencebetween the cytolytic CD4+ T-cells with suppressive properties of theinvention and natural CD4+ regulatory T-cells.

To determine whether naïve CD4+ T cells showed increased AKT kinaseactivity when incubated in vitro with peptides containing athioreductase motif, we purified CD4 T cells from splenocytes of naïveC57BL/6 mice expressing a TCR transgene specific for peptide of SEQ IDNO:6. Cells were stimulated once for 15 minutes with T cell depletedsplenocytes preloaded with peptide of SEQ ID NO:6 (FIG. 5, lane 5)containing no thioreductase motif, or with peptide of SEQ ID NO:2 (FIG.5, lane 6) containing a thioreductase motif. Cell lysis and SDS-PAGEelectrophoresis were carried out as described above. The membrane wasprobed with the antibody recognising activated AKT (Ser473).

Densitometric analysis showed that phosphorylation of AKT was 30% higherwhen naïve cells were stimulated with peptide of SEQ ID NO:2 thanphosphorylation obtained with peptide of SEQ ID NO:6. Thus, a singleincubation of naïve CD4 T cells with a peptide containing athioreductase motif is sufficient to elicit significantly higher kinaseactivity of AKT than observed with cells incubated with the same peptidebut with no thioreductase motif.

Example 8 Cytolytic CD4+ T Cells Induce Apoptosis of Antigen-PresentingCells After Cognate Peptide Recognition

Two distinct populations of APCs (WEHI cells) were loaded for 1 hr witheither peptide of SEQ ID NO:1 or with peptide of SEQ ID NO:9. The cellsloaded with peptide of SEQ ID NO:1 were labelled with 80 nM CFSE; thoseloaded with peptide of SEQ ID NO:9 were labelled with 300 nM CFSE. Thetwo APC populations could therewith be distinguished from each other.CFSE is a label for cytoplasmic proteins enabling the follow-up of celldivisions based on staining intensity, which is reduced by 50% afterevery cell division, but also the identification of a cell populationwithin complex mixtures of cells in culture. CFSE-labelled cells weremixed and subsequently incubated for 18 hrs with a cytolytic CD4 T cellclone (G121) obtained by immunisation with peptide of SEQ ID NO:1.Peptide p71-85 (SEQ ID NO:9, Der p2*−) represents an alternative majorT-cell epitope derived from Der p 2 but does not comprise athioreductase-active motif. Apoptosis of CFSE-labelled APCs was measuredby the binding of annexin V. WEHI cells presenting p21-35 were fullylysed, whereas only about 40% of p71-85(SEQ ID NO:1, Der p2)-loadedcells were affected. As a control, unloaded APCs were used. Results aredepicted in FIG. 6A.

FIG. 6B shows that splenic B cells from naïve C57BU6 mice were inducedinto apoptosis when cultured for 18 hrs with a cytolytic CD4 T cellclone obtained from mice immunised with peptide of SEQ ID NO:2, as shownby dual staining with annexin V and 7-AAD (FIG. 6B, lower panel) but notwhen the antigen was absent (FIG. 6B, upper panel).

FIG. 6C shows the killing of CFSE-stained WEHI B cells loaded withpeptide of SEQ ID NO:11 (TYLRLVKIN, a T epitope derived from the HSV-1virus) and co-cultured for 18 hrs with a cell line obtained from miceimmunised with peptide of SEQ ID NO:12 (CGHCTYLRLVKIN), which comprisesa thioreductase motif. More than 65% of the WEHI cells were stainedpositive for Annexin V (FIG. 6C, lower panel), as opposed to backgroundstaining (19%) obtained when a control T cell line derived from miceimmunised with a peptide of SEQ ID NO:11 was used (FIG. 6C, upperpanel).

Example 9 Cytolytic CD4+ T Cells Induce Apoptosis of Bystander T Cells

The mechanism of bystander T cell suppression was examined withpolyclonal CD4+CD25(−) T cells and with various CD4+ effector T cellclones.

The capacity of cytolytic CD4+ T cells to suppress the proliferation ofCFSE labelled CD4+CD25(−) T cells activated by incubation with anantibody to CD3 in the presence of antigen-presenting cells was assayed.Two cytolytic CD4+ T cell clones elicited by immunisation with peptideof SEQ ID NO:1 (G121 and R3TB7, respectively; indicated in FIG. 7A as“CD4+ (Der p2) clone”) were used. The APCs were loaded with peptide ofSEQ ID NO:5 (indicated in Figure 7A as “APC (Der p 2+)”). The number ofdetectable CD4+CD25(−) T cells (frames), as well as the number ofobserved divisions dramatically dropped within 48 h incubation wheneither one of the two cytolytic clones were added (FIG. 7A, middle andright panels). Interestingly, only activated CD4+CD25(−) T cells werelysed. The control experiment in which the cytolytic CD4+ T-cells werereplaced by an identical number of unlabeled CD4+CD25(−) T cellseliminated a possible artefact related to variable numbers of totalcells in the culture medium (FIG. 7A, left panel). P1 to P3 in theFigure depict the decrease in CFSE labelling as a function of the numberof cell divisions.

An effector CD4+ T cell clone obtained from BALB/c mice immunised with amajor epitope from the allergen Der p1 (SNYCQIYPPNANKIR, SEQ ID NO:13)was labelled with CFSE and incubated with APC loaded with the samepeptide (114-128). When an identical number of unlabelled effector cellswas added, a 40% baseline mortality of the CFSE-labelled cells wasobserved. When co-cultured with a cytolytic CD4 cell clone obtained frommice immunised with peptide of SEQ ID NO:8, more than 73% of theeffector T cells died.

Similar results were obtained when a CD4 T cell line obtained from miceimmunised with natural epitope from ovalbumin (ISQAVHAAHAEINEAGR, SEQ IDNO:14) was used as a bystander target for apoptosis. The cell line waslabelled with CFSE (denoted in FIG. 7B as “labelled CD4+ (OVA−)”) andcultured with peptide of SEQ ID NO:14-loaded splenic APC (denoted inFIG. 7B as “APC (OVA−)”) followed by staining with apoptosis markersannexin V and 7-AAD.

FIG. 7B shows that 27% or 24% of the bystander CD4 cells were alive(annexin V and 7-AAD negative) when cultured with APC alone (left panel)or with APC and the same unlabelled CD4 T cell line (denoted in FIG. 7Bas “unlabeled CD4+ (OVA−)”; right panel of FIG. 7B), respectively. Whenco-cultured with a T cell line derived from mice immunised with theovalbumin peptide comprising a thioreductase motif (SEQ ID NO:4; cellline denoted in FIG. 7B as “CD4+ (OVA+)”), less than 1% of the labelledbystander cells were detected within the double-negative regioncorresponding to living cells.

1. An isolated population of cytotoxic CD4+ T-cells characterised, whencompared to natural CD4+ regulatory T-cells, by undetectable expressionof the transcription repressor Foxp3.
 2. The population of cytotoxicCD4+ T-cells according to claim 1, further characterised, when comparedto natural CD4+ regulatory T-cells, by an increased activity of theserine-threonine kinase AKT.
 3. The population of cytotoxic CD4+ T-cellsaccording to claim 1, further characterised, when compared to naturalCD4+ regulatory T-cells, by undetectable production of TGF-beta andundetectable or very low production of IL-10.
 4. The population ofcytotoxic CD4+ T-cells according to claim 1, further characterised, whencompared to natural CD4+ regulatory T-cells, by production of highconcentrations of IFN-gamma.
 5. The population of cytotoxic CD4+ T-cellsaccording to claim 1, fruther characterised, when compared to naturalCD4+ regulatory T-cells, by production of high concentrations of solubleFas ligand (FasL).
 6. An isolated population of cytotoxic CD4+ T-cellscharacterised, when compared to CD4+ effector cells, by co-expression ofthe transcription activators T-bet and GATA3 after antigenicstimulation.
 7. The population of cytotoxic CD4+ T-cells according toclaim 6 further characterised, when compared to CD4+ effector cells, byconstitutive expression of cell surface proteins CD25 and GITR, and ofintracellular CTLA-4.
 8. The population of cytotoxic CD4+ T-cellsaccording to claim 6, further characterised, when compared to CD4+effector cells, by expression of NKG2D.
 9. The population of cytotoxicCD4+ T-cells according to claim 6, or further characterised, whencompared to CD4+ effector cells, by production of high concentrations ofsoluble Fas ligand (FasL).
 10. An isolated population of cytotoxic CD4+T-cells characterised, when compared to NK cells, by expression of theCD4 co-receptor.
 11. The isolated population of cytotoxic CD4+ T-cellsaccording to claim 10 further characterised, when compared to NaturalKiller cells (NK), by undetectable expression of CD49b.
 12. An isolatedpopulation of cytotoxic CD4+ T-cells characterised, when compared toNatural Killer T cells (NKT), by absence of expression of the invariantalpha chain of the T cell receptor.
 13. The isolated population ofcytotoxic CD4+ T-cells according to claim 12 further characterised, whencompared to NKT cells, by expression of re-arranged beta chain of the Tcell receptor.
 14. The isolated population of cytotoxic CD4+ T-cellsaccording to claim 12, or further characterised, when compared to NKTcells, by lack of CD1d restriction.
 15. A method for obtaining in vivoor ex vivo the population of cytotoxic CD4+ T-cells according to claim1, said method comprising the steps of: providing isolated natural naïveor memory CD4+ T-cells; contacting said cells with an immunogenicpeptide comprising a T-cell epitope and, adjacent to said T-cell epitopeor separated therefrom by a linker of at most 7 amino acids, aC-(X)2-[CST] or [CST]-(X)2-C motif; and expanding said cells in thepresence of IL-2.
 16. A method of identifying a population of cytotoxicCD4+ T-cells, said method comprising the steps of: providing isolatednatural CD4+ regulatory T-cells; providing isolated CD4+ regulatoryT-cells suspected of being cytotoxic; and determining that the T-cellsprovided in (ii) display, compared to the T-cells provided in (i),undetectable expression of the transcription repressor Foxp3
 17. Themethod according to claim 16, said method further comprising determiningin step (iii) that the T-cells provided in (ii) display, compared to theT-cells provided in (i), an increased activity of the serine-threoninekinase AKT.
 18. The method according to claim 16, said method furthercomprising determining in step (iii) that the T-cells provided in (ii)display, compared to the T-cells provided in (i), undetectableproduction of TGF-beta and undetectable or very low production of IL-10.19. The method according to claim 16, said method further comprisingdetermining in step (iii) that the T-cells provided in (ii) display,compared to the T-cells provided in (i), production of highconcentrations of IFN-gamma.
 20. The method according to claim 16, saidmethod further comprising determining in step (iii) that the T-cellsprovided in (ii) display, compared to the T-cells provided in (i),production of high concentrations of soluble Fas ligand (FasL).
 21. Amethod of identifying a population of cytotoxic CD4+ T-cells, saidmethod comprising the steps of: providing isolated natural CD4+ effectorT-cells; providing isolated CD4+ effector T-cells suspected of beingcytotoxic; and (iii) determining that the T-cells provided in (ii)display, compared to the T-cells provided in (i), co-expression of thetranscription activators T-bet and GATA3.
 22. The method according toclaim 21, said method further comprising determining in step (iii) thatthe T-cells provided in (ii) display, compared to the T-cells providedin (i) constitutive expression of cell surface proteins CD25 and GITR,and of intracellular CTLA-4.
 23. The method according to claim 21, saidmethod further comprising determining in step (iii) that the T-cellsprovided in (ii) display, compared to the T-cells provided in (i)expression of NKG2D.
 24. The method according to claim 21, said methodfurther comprising determining in step (iii) that the T-cells providedin (ii) display, compared to the T-cells provided in (i) highconcentrations of soluble Fas ligand (FasL).
 25. A method of identifyinga population of cytotoxic CD4+ T-cells, said method comprising the stepsof: (i) providing isolated natural killer (NK) cells; (ii) providingisolated CD4+ regulatory T-cells suspected of being cytotoxic; and (iii)determining that the T-cells provided in (ii) display, compared to theT-cells provided in (i), the CD4 co-receptor.
 26. The method accordingto claim 25, said method further comprising determining in step (iii)that the T-cells provided in (ii) display, compared to the T-cellsprovided in (i), an undetectable expression of CD49b.
 27. A method ofidentifying a population of cytotoxic CD4+ T-cells, said methodcomprising the steps of: (i) providing isolated natural killer T (NKT)cells; (ii) providing isolated CD4+ regulatory T-cells suspected ofbeing cytotoxic; and (iii) determining that the T-cells provided in (ii)display, compared to the T-cells provided in (i), absence of expressionof the invariant alpha chain of the T cell receptor.
 28. The methodaccording to claim 27, said method further comprising determining instep (iii) that the T-cells provided in (ii) display, compared to theT-cells provided in (i), expression of re-arranged beta chain of the Tcell receptor.
 29. The method according to claim 27, said method furthercomprising determining in step (iii) that the T-cells provided in (ii)display, compared to the T-cells provided in (i), a lack of CD1drestriction.